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Biochemistry 2002, 41, 5462-5472
Mispairing of the 8,9-Dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 Adduct with Deoxyadenosine Results in Extrusion of the Mismatched dA toward the Major Groove† Indrajit Giri, David S. Johnston, and Michael P. Stone* Department of Chemistry and Center in Molecular Toxicology, Vanderbilt UniVersity, NashVille, Tennessee 37235 ReceiVed December 4, 2001; ReVised Manuscript ReceiVed February 11, 2002
ABSTRACT:
The G f T transversion is the dominant mutation induced by the cationic trans-8,9-dihydro8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct. The structure of d(ACATCAFBGATCT)‚d(AGATAGATGT), in which the cationic adduct was mismatched with deoxyadenosine, was refined using molecular dynamics calculations restrained by NOE data and dihedral restraints obtained from NMR spectroscopy. Restrained molecular dynamics calculations refined structures with pairwise rmsd 0.2 Å in entire duplex improper angle (deg) pairwise rmsd (Å) over all atomsa 〈rMDA〉 vs 〈rMDB〉
329 181 110 38 15 93 33 0.05 ( 0.001 8(2 0.33 ( 0.03 1.05 ( 0.15
〈rMDA〉, average of 10 converged structures starting from A-DNA; 〈rMDB〉, average of 10 converged structures from B-DNA. a
the AFBG6‚A15 mismatched base pair. The restraints used for rMD calculations are listed in Table S4 of the Supporting Information. Calculations begun from IniA and IniB structures generated by INSIGHTII yielded 10 “converged” structures (Figure 5). The maximum pairwise rmsd for the converged structures was 0.55 Å, indicating a well-defined conformation. A further 1.4 ns rMD simulation in the presence of solvent and counterions was performed using the AMBER force field. The maximum pairwise rmsd for the structures emergent from the AMBER calculations was 1.2 Å. Sixth root residual factors R1x calculated from complete relaxation matrix analysis using CORMA (64) are collected in Table 3. The starting structure IniA did not provide a satisfactory fit for the CORMA calculations. The IniB starting structure provided a better fit, suggesting that the actual conformation of the adducted duplex was closer to B-form DNA than to A-form DNA. Nevertheless, the R1x values arising from comparison of the IniB structure with the experimental NOEs were greater than 15%, suggesting that the AFB1 adduct perturbed the duplex structure at the lesion site. The refined structure gave R1x values in the range of 10.5-11.6% as a function of NOE mixing time, with the best agreement shown to the 150 ms mixing time NOE data. Overall, the relaxation matrix calculations suggested that the refined structures provided reasonable models for the adducted duplex. Structural EValuation. The refined structure was a righthanded duplex (Figure 6). Figures 7 and 8 show detailed views of the adduct site. The duplex suffered localized distortion at and immediately adjacent to the adduct site, evidenced by the increased rise of 7.7 Å as compared to the
5468 Biochemistry, Vol. 41, No. 17, 2002
Giri et al.
FIGURE 5: Superposition of 10 structures emergent from rMD calculations of the AFB1-modified mismatched duplex. The large number of NOEs observed between the AFB1 moiety and the DNA resulted in excellent convergence of the calculations at and adjacent to the lesion site. Table 3: Sixth Root Residual Indices R1x as a Function of NOE Mixing Timea,b structure rMD final rMDBd IniBe
c
120 ms
150 ms
200 ms
11.6 11.9 16.5
10.5 10.8 15.1
10.9 11.7 19.6
a All values for R x are ×10-2. To exclude end effects, only the eight 1 inner base pairs were included in calculations. b R1x ) ∑|(ao)i1/6 (ac)i1/6|/∑|(ao)i1/6|, where (ao) and (ac) are the intensities of observed (nonzero) and calculated NOE cross-peaks. c Calculated for the structure emergent from rMD calculations in the presence of counterions and solvent. d Calculated for the structure emergent from rMD calculations in vacuo. e Calculated from the B-form starting structure.
value of 3.5 Å normally observed for B-DNA between mismatch AFBG6‚A15 and C5‚G16. These two base pairs buckled in opposite directions away from the intercalated aflatoxin moiety. Changes of 24° and -14° in buckle were calculated for C5‚G16 and AFBG6‚A15, respectively, similar to what was observed in crystallographically determined intercalation structures (82, 83). Unwinding of the duplex was observed. The helical twist at the intercalation site C5‚G16 f AFBG6‚A15 was reduced to ∼8.1°, as compared to the expectation value of ∼34°. This was also evident from the 55° base pair opening value for the mismatched AFBG6‚A15 pair. The rMD calculations predicted that A15 was not hydrogen bonded to AFBG6 but rather was shifted toward the major groove. Helicoidal analysis showed that with the exception of base pair step 5, C5‚G16 f AFBG6‚A15, interbase pair parameters converged to values consistent with a right-
FIGURE 6: Stick and ribbon model showing the average structure of the mismatched AFB1-modified duplex predicted from rMD calculations. The modified nucleotide AFBG6 is shown in magenta. The mismatched A15 is shown in yellow. One consequence of AFB1 intercalation 5′ to the modified deoxyguanosine was the increased rise and unwinding of the duplex at the lesion site.
handed B-like helix. Larger deviations were found for base pair shearing, stretch, rise, propeller twist, and opening near and at the adduct site. The helicoidal analysis is detailed in Figure S3 of the Supporting Information. DISCUSSION In DNA, mismatches arise through errors in replication or through recombination processes. If not repaired, they lead to mutations in the genome. Thus, their recognition and the efficiency of their repair are of considerable interest. The G f T transversion is the predominant mutation introduced by aflatoxin B1 (3, 30). Presumably, this mutation occurs as a consequence of the incorrect incorporation of dATP opposite the AFBG lesion during DNA replication. Sitespecific mutagenesis data carried out in a bacterial system supported the notion that the trans-8,9-dihydro-8-(N7guanyl)-9-hydroxy-aflatoxin B1 lesion was responsible for G‚C f T‚A transversions (31). The observation that the trans-8,9-dihydro-8-(N7-guanyl)-9-hydroxy-aflatoxin B1 adduct allowed correct incorporation of cytosine by DNA polymerase I (exo-) opposite but resulted in polymerase blockage, while incorrect incorporation of adenine allowed full-length extension (40), led to further interest in the structure of the adduct mismatched with deoxyadenosine. The AFBG‚A Mismatched Oligodeoxynucleotide. In the mismatched oligodeoxynucleotide, the aflatoxin moiety intercalated above the 5′ face of the modified guanine, such that the aflatoxin methoxy and cyclopentenone ring protons faced into the minor groove, whereas the furofuran ring protons faced into the major groove (Figure 7). The overall
Aflatoxin B1 Opposite a Mismatched Adenine
Biochemistry, Vol. 41, No. 17, 2002 5469
FIGURE 8: Comparison of the mismatched AFBG‚A structure with the correctly paired AFBG‚C structure (35). Stacking of the adducted base pair and the 3′ flanking bases as predicted from rMD calculations. The adducted nucleotide AFBG6 is shown in magenta. The 3′-neighbor base pair A7‚T14 is shown in black. (A) Mismatched AFBG‚A structure. The mismatched nucleotide A15 is shown in yellow and is shifted toward the major groove. (B) Correctly paired AFBG‚C structure. The correctly paired C15 is shown in yellow. FIGURE 7: Detailed view of the mispaired AFBG6‚A15 site and the flanking base pairs C5‚G16 and A7‚T14. The adducted nucleotide AFBG6 is shown in magenta. The mismatched A15 is shown in green. Protons are shown in yellow. (A) View from the major groove. The AFB1 H6a, H8, H9, and H9a protons face the major groove. (B) View from the minor groove. The AFB1 -OCH3 and H5 protons face the minor groove, as do the AFB1 methylene protons H2 R,β and H3 R,β.
conformation of the DNA remained right-handed, and the principal perturbation to the DNA structure occurred at the site of the mismatched AFBG6‚A15 base pair. The intercalation of the AFB1 moiety on the 5′ side of the adducted dG was reminiscent of other aflatoxin-adducted oligodeoxynucleotides (32, 34, 36). The AFB1 protons had nearly identical chemical shifts and also exhibited the same pattern of NOEs to the 5′-flanking base in all cases. This motif appears to be a characteristic of the trans-8,9-dihydro-8-(N7-guanyl)-9hydroxy-aflatoxin B1 adduct which is conserved irrespective of the nature of the base complementary to the adducted dG. The refined structure was supported by NOE evidence. The NOESY connectivities through the adducted strand were disrupted by the presence of the aflatoxin lesion similar to that reported before (36). A number of characteristic NOEs were observed between the aflatoxin moiety and the 5′neighbor base pair, C5‚G16. The aflatoxin H6a and H9a protons, facing the major groove, exhibited NOEs to C5 H5. AFB1 H6a also showed an NOE to C5 H6. The observations of NOEs between the AFB1 4-OCH3 and H5 protons to C5 H1′ and AFBG6 H1′, and the NOE between the 4-OCH3 protons and AFBG6 H4′, were consistent with intercalation. These oligodeoxynucleotide protons faced into the minor groove and suggested that the AFB1 moiety spanned the helix. The presence of the AFB1 moiety resulted in upfield chemical shifts for the protons of the AFB1 moiety as
compared to unbound aflatoxin. The pattern and magnitude of these chemical shift changes were supportive of the notion that the orientation of the covalently bound aflatoxin moiety was similar to previously observed structures (36). The observations that the intensity of the A15 H8 to A15 H1′ cross-peak was smaller than cytosine H5-H6 crosspeaks and that A15 H2 exhibited a typical value for its chemical shift of 7.88 ppm supported the conclusion that the mismatched A15 was in the anti conformation about the glyosyl bond. This conclusion was also consistent with the observed NOEs from the A15 H2 proton to the aflatoxin protons. The cross-peak between A15 H2 and G16 H1′ also confirmed the glycosyl conformation of the mismatched adenine A15. These would not have been consistent with the syn conformation. The conclusion that A15 was not hydrogen bonded to AFBG6 but rather was shifted toward the major groove (Figure 8) was consistent with the observation that the AFBG6 N1H proton was not observed. The delocalization of the positive charge induced by alkylation of the guanine N7 position through the adducted base and aflatoxin moiety (36) was anticipated to cause broadening of the AFBG6 N1H proton. The downfield shift of AFBG6 H8 (Figure 1) was due to the positive charge on the imidazole ring of the modified guanine, which also increased the rate of exchange with solvent, rendering this proton resonance unobservable in the deuterated buffer. However, the AFBG N1H proton was observed in the correctly paired AFBG‚C adduct as a broadened signal shifted downfield in the spectrum (24). Thus, the failure to observe the AFBG6 N1H proton in the AFBG6‚A15 pairing interaction was consistent with the notion that it did not participate in hydrogen bonding and was exchange broadened. Further support for the predicted conformation in which A15 was shifted toward the major
5470 Biochemistry, Vol. 41, No. 17, 2002 groove was provided by rMD calculations in which specific potential base pairing restraints were incorporated between AFB 6 G and A15. In each instance, rMD calculations carried out with such restraints resulted in significant NOE violations when compared with the spectroscopic data. Additional evidence in support of the calculated structure involving A15 accrued from chemical shift perturbations at base pair A7‚ T14. That normal base pairing was present at A7‚T14 was evident from the cross-peaks between T14 N3H with T8 N3H and A7 H2, respectively. Comparison to the Properly Paired AFBG‚C Duplex. The conformation of the mismatched dA in the AFBG‚A mismatched duplex was different as compared to dC in the correctly paired AFBG‚C modified duplex (Figure 8) (35). Misincorporation of adenine opposite AFBG6 resulted in greater unwinding of the helix compared to the AFBG‚C context. For AFBG‚C, both the lesion site and 3′-flanking pair were in a position to base pair. The presence of base pairing was suggested by the observance of the AFBG N1H resonance, which underwent sufficiently slow exchange with solvent to be observable. This was predicted by the rMD calculations of the two helices. For the AFBG‚C helix, the top view suggested that C15 is nicely stacked above T14. In contrast, for the AFBG‚A context (Figure 8), the calculations predicted A15 shifted toward the major groove, in an orientation in which it could not participate in hydrogen bonding with AFBG6. For the AFBG‚A context the mismatched adenine was more stacked on the deoxyribose of T14 than above the nucleobase of T14. This perhaps explained the unusual chemical shift effects observed for T14. This orientation of the dA base in the AFBG‚A duplex, moved away from the cationic adduct compared to AFBG‚C, might provide a rationale for the observation that when dA was incorrectly inserted opposite AFBG, DNA polymerase I exo- successfully bypassed the adduct and continued replication (40). Effect of the Aflatoxin B1 Adduct on the G‚A Mismatch. The results suggested AFB1 stabilized a single conformation of the AFBG6‚A15 base pair at neutral pH. The observed pattern of NOEs led to the conclusion that the glycosyl bond of A15 remained in the anti conformation. The corresponding duplex containing G6‚A15, but lacking the AFB1 adduct, exhibited spectral line broadening at neutral pH. This was interpreted to result from intermediate conformational exchange, possibly similar to that reported by Patel and co-workers (48). The possibility of sheared base pairing associated with tandem G‚A mismatches (41, 49) was ruled out. Sheared pairing was not expected because the AFBG6‚A15 mismatch was incorporated into a nontandem sequence context. The absence of downfield 31P shifts in the present instance was consistent with this conclusion (42, 44). The results provide additional insight into the structure of a G‚A mismatch in the presence of a DNA lesion. The AFBG‚A mismatch structure differed from G‚A mismatches in the presence of the adenyl N6 PAH adducts (48, 54, 55). The G‚A mismatch pair was stable in the presence of a 10S adduct derived from addition of the dA N6 amino group to (+)-(7R,8S,9S,10R)7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. Two conformations of the mismatch in the presence of the PAH lesion were attributed to interconversion of the modified dA nucleotide between the syn and anti conformations about the glycosyl bond. In the major conformation, the glycosyl bond of the modified dA was in the syn
Giri et al. conformation, resulting in the projection of the modified dA more into the major groove (54-56). The G‚A mismatch pair was also examined in the presence of a 10R adduct derived from addition of the dA N6 amino group to (-)(7S,8R,9R,10S)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene. The mismatched dA remained in the anti conformation about the glycosyl bond, with the dG being pushed into the major groove (54). Biological Significance. Experiments using DNA polymerase I exo- in vitro showed that replication was blocked following correct insertion of dC opposite AFBG, but successful bypass occurred following incorrect incorporation of dA (40). The unmodified mismatched duplex appeared to be in a disordered state consisting of a blend of conformations. In contrast, the mispaired dA in the modified AFBG‚A mismatch existed in a single conformation. The glycosyl bond of the mismatched dA was in the anti conformation. This was consistent with the observation that the oligodeoxynucleotide containing the AFBG‚A mismatch had a lower melting temperature than did the oligodeoxynucleotide containing the correct AFBG‚C pair. Our working hypothesis posits that the different structure of the AFBG‚A mismatch pair as compared to the proper AFBG‚C pair perhaps facilitates replication bypass of the AFB1 lesion by DNA polymerase I exo- (40) following adventitious misincorporation of dA opposite the lesion. ACKNOWLEDGMENT We thank Dr. Zhijun Li for assistance with rMD calculations and Mr. Markus Voehler for assistance with NMR spectroscopy. SUPPORTING INFORMATION AVAILABLE Tables S1-S3, which detail the 1H NMR chemical shift assignments, and S4, which shows the experimental distances and classes of restraints; Figures S1, which shows atomic names and types used in AMBER calculations for AFB1 and the partial charges, S2, which shows the expanded sequential NOE connectivities for the unmodified G‚A mismatched duplex, and S3, which shows helicoidal analysis of the refined structure. REFERENCES 1. Busby, W. F., Jr., and Wogan, G. N. (1984) in Chemical Carcinogens (Searle, C. E., Ed.) pp 945-1136, American Chemical Society, Washington, DC. 2. McCann, J., Spingarn, N. E., Koburi, J., and Ames, B. N. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 979-983. 3. Foster, P. L., Eisenstadt, E., and Miller, J. H. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2695-2698. 4. Foster, P. L., Groopman, J. D., and Eisenstadt, E. (1988) J. Bacteriol. 170, 3415-3420. 5. Bailey, G. S., Loveland, P. M., Pereira, C., Pierce, D., Hendricks, J. D., and Groopman, J. D. (1994) Mutat. Res. 313, 25-38. 6. Bailey, G. S., Williams, D. E., Wilcox, J. S., Loveland, P. M., Coulombe, R. A., and Hendricks, J. D. (1988) Carcinogenesis 9, 1919-1926. 7. McMahon, G., Davis, E. F., Huber, L. J., Kim, Y., and Wogan, G. N. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 1104-1108. 8. Soman, N. R., and Wogan, G. N. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 2045-2049. 9. Bressac, B., Kew, M., Wands, J., and Ozturk, M. (1991) Nature 350, 429-431.
Aflatoxin B1 Opposite a Mismatched Adenine 10. Hsu, I. C., Metcalf, R. A., Sun, T., Welsh, J. A., Wang, N. J., and Harris, C. C. (1991) Nature 350, 427-428. 11. Greenblatt, M. S., Bennett, W. P., Hollstein, M., and Harris, C. C. (1994) Cancer Res. 54, 4855-4878. 12. Shen, H. M., and Ong, C. N. (1996) Mutat. Res. 366, 23-44. 13. Soini, Y., Chia, S. C., Bennett, W. P., Groopman, J. D., Wang, J. S., DeBenedetti, V. M., Cawley, H., Welsh, J. A., Hansen, C., Bergasa, N. V., Jones, E. A., DiBisceglie, A. M., Trivers, G. E., Sandoval, C. A., Calderon, I. E., Munoz Espinosa, L. E., and Harris, C. C. (1996) Carcinogenesis 17, 1007-1012. 14. Lunn, R. M., Zhang, Y. J., Wang, L. Y., Chen, C. J., Lee, P. H., Lee, C. S., Tsai, W. Y., and Santella, R. M. (1997) Cancer Res. 57, 3471-3477. 15. Mace, K., Aguilar, F., Wang, J. S., Vautravers, P., GomezLechon, M., Gonzalez, F. J., Groopman, J., Harris, C. C., and Pfeifer, A. M. (1997) Carcinogenesis 18, 1291-1297. 16. Santella, R. M., Zhang, Y. J., Chen, C. J., Hsieh, L. L., Lee, C. S., Haghighi, B., Yang, G. Y., Wang, L. W., and Feitelson, M. (1993) EnViron. Health Perspect. 99, 199-202. 17. Chen, C. J., Yu, M. W., Liaw, Y. F., Wang, L. W., Chiamprasert, S., Matin, F., Hirvonen, A., Bell, D. A., and Santella, R. M. (1996) Am. J. Hum. Genet. 59, 128-134. 18. Yu, M. W., Lien, J. P., Chiu, Y. H., Santella, R. M., Liaw, Y. F., and Chen, C. J. (1997) J. Hepatol. 27, 320-330. 19. Ueng, Y. F., Shimada, T., Yamazaki, H., and Guengerich, F. P. (1995) Chem. Res. Toxicol. 8, 218-225. 20. Baertschi, S. W., Raney, K. D., Stone, M. P., and Harris, T. M. (1988) J. Am. Chem. Soc. 110, 7929-7931. 21. Johnson, W. W., Harris, T. M., and Guengerich, F. P. (1996) J. Am. Chem. Soc. 118, 8213-8220. 22. Essigmann, J. M., Croy, R. G., Nadzan, A. M., Busby, W. F., Jr., Reinhold, V. N., Buchi, G., and Wogan, G. N. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1870-1874. 23. Stone, M. P., Gopalakrishnan, S., Harris, T. M., and Graves, D. E. (1988) J. Biomol. Struct. Dyn. 5, 1025-1041. 24. Gopalakrishnan, S., Stone, M. P., and Harris, T. M. (1989) J. Am. Chem. Soc. 111, 7232-7239. 25. Gopalakrishnan, S., Byrd, S., Stone, M. P., and Harris, T. M. (1989) Biochemistry 28, 726-734. 26. Stone, M. P., Gopalakrishnan, S., Raney, K. D., Raney, V. M., Byrd, S., and Harris, T. M. (1990) in Molecular Basis of Specificity in Nucleic Acid-Drug Interactions (Pullman, B., and Jortner, J., Eds.) pp 451-480, Kluwer Academic Publishers, Dordrecht, The Netherlands. 27. Raney, K. D., Gopalakrishnan, S., Byrd, S., Stone, M. P., and Harris, T. M. (1990) Chem. Res. Toxicol. 3, 254-261. 28. Raney, V. M., Harris, T. M., and Stone, M. P. (1993) Chem. Res. Toxicol. 6, 64-68. 29. Iyer, R. S., Coles, B. F., Raney, K. D., Thier, R., Guengerich, F. P., and Harris, T. M. (1994) J. Am. Chem. Soc. 116, 16031609. 30. Bailey, E. A., Iyer, R. S., Harris, T. M., and Essigmann, J. M. (1996) Nucleic Acids Res. 24, 2821-2828. 31. Bailey, E. A., Iyer, R. S., Stone, M. P., Harris, T. M., and Essigmann, J. M. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 1535-1539. 32. Gopalakrishnan, S., Harris, T. M., and Stone, M. P. (1990) Biochemistry 29, 10438-10448. 33. Gopalakrishnan, S., Liu, X., and Patel, D. J. (1992) Biochemistry 31, 10790-10801. 34. Jones, W. R., Johnston, D. S., and Stone, M. P. (1998) Chem. Res. Toxicol. 11, 873-881. 35. Giri, I., Jenkins, M. D., Schnetz-Boutaud, N. C., and Stone, M. P. (2002) Chem. Res. Toxicol. (in press). 36. Johnston, D. S., and Stone, M. P. (1995) Biochemistry 34, 14037-14050. 37. Green, C. L., Loechler, E. L., Fowler, K. W., and Essigmann, J. M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 13-17. 38. Loechler, E. L., Green, C. L., and Essigmann, J. M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6271-6275. 39. Basu, A. K., and Essigmann, J. M. (1988) Chem. Res. Toxicol. 1, 1-18. 40. Johnston, D. S., and Stone, M. P. (2000) Chem. Res. Toxicol. 13, 1158-1164.
Biochemistry, Vol. 41, No. 17, 2002 5471 41. Cheng, J. W., Chou, S. H., and Reid, B. R. (1992) J. Mol. Biol. 228, 1037-1041. 42. Nikonowicz, E. P., Meadows, R. P., Fagan, P., and Gorenstein, D. G. (1991) Biochemistry 30, 1323-1334. 43. Chou, S. H., Cheng, J. W., and Reid, B. R. (1992) J. Mol. Biol. 228, 138-155. 44. Nikonowicz, E. P., and Gorenstein, D. G. (1990) Biochemistry 29, 8845-8858. 45. Kan, L. S., Chandrasegaran, S., Pulford, S. M., and Miller, P. S. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4263-4265. 46. Patel, D. J., Kozlowski, S. A., Ikuta, S., and Itakura, K. (1984) Biochemistry 23, 3207-3217. 47. Greene, K. L., Jones, R. L., Li, Y., Robinson, H., Wang, A. H., Zon, G., and Wilson, W. D. (1994) Biochemistry 33, 1053-1062. 48. Gao, X., and Patel, D. J. (1988) J. Am. Chem. Soc. 110, 51785182. 49. Li, Y., Zon, G., and Wilson, W. D. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 26-30. 50. Maskos, K., Gunn, B. M., LeBlanc, D. A., and Morden, K. M. (1993) Biochemistry 32, 3583-3595. 51. Brown, T., Leonard, G. A., Booth, E. D., and Chambers, J. (1989) J. Mol. Biol. 207, 455-457. 52. Brown, T., Hunter, W. N., Kneale, G., and Kennard, O. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 2402-2406. 53. Hunter, W. N., Brown, T., and Kennard, O. (1986) J. Biomol. Struct. Dyn. 4, 173-191. 54. Schurter, E. J., Yeh, H. J. C., Sayer, J. M., Lakshman, M. K., Yagi, H., Jerina, D. M., and Gorenstein, D. G. (1995) Biochemistry 34, 1364-1375. 55. Yeh, H. J. C., Sayer, J. M., Liu, X., Altieri, A. S., Byrd, R. A., Lakshman, M. K., Yagi, H., Schurter, E. J., Gorenstein, D. G., and Jerina, D. M. (1995) Biochemistry 34, 1357013581. 56. Schwartz, J. L., Rice, J. S., Luxon, B. A., Sayer, J. M., Xie, G., Yeh, H. J., Liu, X., Jerina, D. M., and Gorenstein, D. G. (1997) Biochemistry 36, 11069-11076. 57. Murray, R. W., and Jeyaraman, R. (1985) J. Org. Chem. 50, 2847-2853. 58. Piotto, M., Saudek, V., and Sklenar, V. (1992) J. Mol. Biol. 6, 661-665. 59. Arnott, S., and Hukins, D. W. L. (1972) Biochem. Biophys. Res. Commun. 47, 1504-1509. 60. Arnott, S., and Hukins, D. W. L. (1973) J. Mol. Biol. 81, 93105. 61. Frisch, M. J., Trucks, G. W., et al. (1998) Gaussian 98, Gaussian Inc., Pittsburgh, PA. 62. Bayly, C. I., Cieplak, P., Cornell, W. D., and Kollman, P. A. (1993) J. Phys. Chem. 40, 10269-10280. 63. Borgias, B. A., and James, T. L. (1990) J. Magn. Reson. 87, 475-487. 64. Keepers, J. W., and James, T. L. (1984) J. Magn. Reson. 57, 404-426. 65. James, T. L. (1991) Curr. Opin. Struct. Biol. 1, 1042-1053. 66. Brunger, A. T. (1992) in X-PLOR Version 3.1. A system for X-ray Crystallography and NMR, Yale University Press, New Haven, CT. 67. Clore, G. M., Gronenborn, A. M., Carlson, G., and Meyer, E. F. (1986) J. Mol. Biol. 190, 259-267. 68. Ryckaert, J.-P., Ciccotti, G., and Berendsen, H. J. C. (1977) J. Comput. Phys. 23, 327-341. 69. Jorgensen, W. L., Chandrasekhar, J., Madura, J. D., Impey, R. W., and Klein, M. L. (1983) J. Chem. Phys. 79, 926-935.
5472 Biochemistry, Vol. 41, No. 17, 2002 70. Case, D. A., Pearlman, C. A., et al. (1999) AMBER 6.0, University of California, San Francisco, CA. 71. Cornell, W. D., Cieplak, P., Bayly, C. I., Gould, I. R., Merz, K. M., Ferguson, D. M., Spellmeyer, D. C., Fox, T., Caldwell, J. W., and Kollman, P. A. (1995) J. Am. Chem. Soc. 117, 5179. 72. Darden, T., York, D., and Pedersen, L. (1993) J. Chem. Phys. 12, 10089-10092. 73. Essmann, U., Perera, L., Berkowitz, M. L., Darden, T., Lee, H., and Pedersen, L. G. (1995) J. Chem. Phys. 103, 85778593. 74. Allain, F. H., and Varani, G. (1997) J. Mol. Biol. 267, 338351. 75. Mauffret, O., Amir-Aslani, A., Maroun, R. G., Monnot, M., Lescot, E., and Fermandjian, S. (1998) J. Mol. Biol. 283, 643655. 76. Lu, X. J., Shakked, Z., and Olson, W. K. (2000) J. Mol. Biol. 300, 819-840. 77. Reid, B. R. (1987) Q. ReV. Biophys. 20, 2-28.
Giri et al. 78. Patel, D. J., Shapiro, L., and Hare, D. (1987) Q. ReV. Biophys. 20, 35-112. 79. Boelens, R., Scheek, R. M., Dijkstra, K., and Kaptein, R. (1985) J. Magn. Reson. 62, 378-386. 80. Rinkel, L. J., and Altona, C. (1987) J. Biomol. Struct. Dyn. 4, 621-649. 81. Rein, R., Shibata, M., Garduno-Juarez, R., and KieberEmmons, T. (1983) in Structure and Dynamics: Nucleic Acids and Proteins (Clementi, E., and Sarma, R. H., Eds.) pp 269288, Adenine Press, New York. 82. Egli, M., Williams, L. D., Frederick, C. A., and Rich, A. (1991) Biochemistry 30, 1364-1372. 83. Frederick, C. A., Williams, L. D., Ughetto, G., Van der Marel, G. A., Van Boom, J. H., Rich, A., and Wang, A. H. J. (1990) Biochemistry 29, 2538-2549. BI012116T
